Work on the effects of the urea series of denaturants on the conformation and subunit dissociation of human hemoglobin is being continued, using absorption, optical rotatory dispersion, circular dichroism, osmometric and light scattering methods. Light scattering measurements have confirmed our observations based on earlier osmotic pressure measurements, namely that the urea series of solutes cause increasing dissociation of hemoglobin tetramer to alpha-beta half- molecules, with increasing chain length and hydrophobicity of the urea. The order of increasing effectiveness of the ureas as subunit dissociating agents (at low urea concentration where essentially no denaturation is observed) and denaturants is found to be the same; that is, urea methylurea ethylurea propylurea butylurea. The single-chain hemoglobin isolated from the blood worm, Glycera dibranchiata is found to have nearly the same denaturation midpoints as the four chain human hemoglobin (both studied in the CO stabilized, Fe2 ion state). Analysis of the denaturation data, based on the theories of Peller and Flory with appropriate binding constants, KB, assuming group additivity of the urea KB's (with a hydrophobic-(CH2)n contribution and a polar -HN (CO) NH2 contribution) gave satisfactory prediction of the effects of increasing hydrocarbon content. As in the case of other proteins studied in our laboratory, it is found that hydrophobic interactions play a progressingly greater role with increasing hydrocarbon content in the mode of action of the urea and also the amide series of denaturants.